Doctor of Philosophy
Dr. John Lewis
The development of screening approaches to identify novel affinity ligands has paved the way for a new generation of molecular targeted nanomedicines. To identify novel targeting ligands, several studies have demonstrated the advantages in screening one-bead-one-compound (OBOC) libraries. Conventional methods typically bias the display of the target protein to ligands during the screening process. We have developed an unbiased multiplex ‘beads on a bead’ strategy to isolate, characterize, and validate high affinity ligands from OBOC libraries. In addition, due to the advantages associated with screening OBOC libraries directly against living cells, we sought to combine cell-based screen methods with automated high-throughput technologies to facilitate the identification of novel affinity ligands. We have shown that bound cells can be reversibly cross-linked onto the beads, and then easily sorted through automated means using the Complex Object Parametric Analyzer and Sorter (COPAS) large format flow cytometer (purchased from Union Biometrica) without affecting the sequence deconvolution of peptides using matrix-assisted laser desorption/ionization-time of flight MALDI-TOF mass spectrometry (MS). This high throughput strategy can accelerate the discovery and generation of new targeting agents.
Using the ‘beads on a bead’ approach, we have discovered novel peptides that do not contain the Arg-Gly-Asp (RGD) motif that bind αvβ3 integrin without affecting the biology of cancer or endothelial cells. The peptides identified here represent novel targeting agents for integrins that can be applied to cancer imaging without the risk of increased tumor invasion and metastasis. In order to target angiogenesis, we used the ‘beads on a bead’ strategy again to screen an OBOC library to isolate novel high-affinity peptides against EGFL7. A high-affinity peptide ligand, E7-p72 was shown to target cancer cells and endothelial cells in an EGFL7-dependent manner. This lead candidate could provide a basis for a new generation of sensitive angiogenesis targeting agents for imaging early cancers or delivery of therapeutics to disease sites.
The expansion of our understanding of EGFL7 has also led us to identify and design bioactive peptides from the sequence of EGFL7 that could interfere with its function and serve as angiogenesis inhibitors. Particularly, one peptide, E7-C13 derived from the C-terminus of EGFL7 inhibits angiogenesis. This peptide could provide a basis for a new generation of therapeutic agents for locally advanced cancers.
Cho, Choi-Fong, "Peptide-Mediated Targeting of Angiogenesis for Molecular Imaging and Treatment of Cancer" (2012). Electronic Thesis and Dissertation Repository. 1082.